Gene targeting by means of homologous DNA recombination is a powerful technique for elucidating the function of eukaryotic genes. Depending upon the precise structure and mode of presentation of the added DNA, targeting can result in complete inactivation of the chromosomal target gene or, alternatively, can alter the target's structure or expression in subtle and well-defined ways (Capecchi, M. R. (1989) Science 244, 1288-1292). A key distinction in gene targeting is whether the vector and approach used are intended to cause gene replacement or gene insertion (Hasty, P. et al. (1991) Mol. Cell. Biol. 11, 4509-4517). In the first case, two homologous recombination events replace the region under study with a selectable gene (FIG. 1B). In gene insertion, a single recombination event adds the entire foreign DNA molecule to the targeted locus (FIG. 1C). Gene insertion may be accomplished using either a circular plasmid or one which contains a double strand break within the region of homology, and gene replacement utilizes molecules linearized at a point outside of the homologous regions.
Despite the distinctions drawn between mechanisms of gene replacement and insertion, experiments intended to produce gene replacement often result instead, in a variable proportion of cases, in gene insertion (Hasty, P. et al. (1991) Mol. Cell. Biol. 11, 4509-4517; Thomas, K. R. et al. (1992) Mol. Cell. Biol. 12, 2919-2923; Zhang, H. et al. (1994) Mol. Cell. Biol. 14, 2404-2410). A locus insertionally targeted may, depending upon the vector design, fail to be inactivated; the insertion event is also subject to reversion (Thomas, K. R. et al. (1992) Mol. Cell. Biol. 12, 2919-2923; Hasty, P. et al. (1991) Nature 350, 243-246). Unintended insertional targeting occurs during the transformation of mammalian cells and of the simple eukaryote Dictyostelium discoideum (Manstein, D. J. et al. (1989) EMBO J. 8, 923-932; Sun, T. J. et al. (1991) Genes Dev. 5, 572-582). Events of this type are thought to occur via the joining in vivo of the linearized ends of the plasmid, recreating a circular vector capable of single-crossover insertion at the homologous locus. There is evidence of prolific end-to-end joining activity (i.e., DNA ligation) in a wide variety of eukaryotes. Ligation in vivo occurs whether the ends introduced are "sticky," blunt, or even incompatible (Goedecke, W. et al. (1994) Nucleic Acids Res. 22, 2094-2101; Katz, K. et al. (1990) Ph.D. thesis, University of Massachusetts). It would be desirable to prevent unwanted end-to-end joining of transfecting molecules, and thereby gene insertion, to promote only the desired double cross-over replacement events.
Chang and Wilson (Chang, X.-B. et al. (1987) Proc. Natl. Acad. Sci., USA 84, 4959-4963) observed that 2'3'dideoxy-blocked DNA ends are unable to be ligated in mammalian cells in vivo. There is no information concerning effects of this biochemical procedure on chromosomal integration or gene targeting.